Ion transport and ligand binding by the Na-K-Cl cotransporter, structure-function studies.

The cation-Cl cotransporters (CCCs) mediate the coupled movement of Na and/or K to that of Cl across the plasmalemma of animal cells. Eight CCCs have been identified to date: two Na-K-Cl cotransporters (NKCC), four K-Cl cotransporters (KCCs), one Na-Cl cotransporter (NCC) and one CCC interacting protein (CIP). All of the NKCCs and KCCs are inhibited by loop diuretics; mercury and other modifying agents are also known to block NKCC-mediated transport. In this work, we have utilized a mutational approach to study the interaction between different substrates and the NKCCs. We relied on the strategy of exchanging domains between functionally distinct carriers (the shark NKCCl and the human NKCCl) to identify residues or group of residues that are involved in the interaction with ions, loop diuretics and Hg. Our results show that the N- and C-termini have no role in determining the species differences in ion transport and bumetanide binding. On the other hand, the interaction between Hg and the NKCCs is found to partially involve the C-terminus through residues that contain available sulfhydryl groups. Within the transmembrane segments, variant residues in the 2nd, 4th and 7th predicted alpha-helices are shown to encode the differences in ion transport between the shark and the human cotransporters. For loop diuretic binding, several regions throughout the central domain appear to be involved. Interestingly, these regions are not the same as those involved in cation or anion transport, and in Hg binding.